Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a new macrocycle library known as rapafucins that was inspired by rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, named Rapaglutin A (RgA). We demonstrated that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4 with an IC 50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA is a new chemical tool to study GLUT function and a promising lead to develop anticancer drugs.